Studies of processing of the Alzheimer -amyloid precursor protein (APP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways the "-secretase" pathway, which generates -amyloid (A1-40/42; ~4 kDa), and the "-secretase" pathway, which generates a smaller fragment, the "p3" peptide (A17-40/42; ~3 kDa). To determine whether similar processing events underlie APP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [35S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa A-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional A beginning at position A(Asp1), whereas both radiosequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began wi th A(Glu11) at the N terminus, rather than A(Leu17) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble APP release and decreased generation of both the 4-kDa A and the 3-kDa N-truncated A. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing A secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant A variant peptides and emphasize the role of protein phosphatases in modulating neuronal A generation. Gunnar K. Gouras, Huaxi Xu, Jasmina N. Jovanovic, Joseph D. Buxbaum, Rong Wang, Paul Greengard, Norman R. Relkin, and Sam Gandy (1998), J. Neurochem. 71, 1920-1925.